Recombinant production of novel polyketides

ABSTRACT

Novel polyketides and novel methods of efficiently producing both new and known polyketides, using recombinant technology, are disclosed. In particular, a novel host-vector system is described which is used to produce polyketide synthases which in turn catalyze the production of a variety of polyketides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/828,898 filed Mar.31, 1997 and now U.S. Pat. No. 6,022,731 which is a continuation of U.S.Ser. No. 08/238,811 filed May 6, 1994 and now U.S. Pat. No. 5,672,491which is a continuation-in-part of U.S. Ser. No. 08/164,301 filed Dec.8, 1993 and now abandoned which is a continuation-in-part of U.S. Ser.No. 08/123,732 filed Sep. 20, 1993 and now abandoned. Priority isclaimed under 35 U.S.C. § 120 and the disclosures of these applicationsare incorporated herein by reference.

REFERENCE TO GOVERNMENT CONTRACT

This invention was made with United States Government support in theform of a grant from the National Science Foundation (BCS-9209901). TheUnited States Government has certain rights to this invention.

TECHNICAL FIELD

The present invention relates generally to polyketides and polyketidesynthases. In particular, the invention pertains to the recombinantproduction of polyketides using a novel host-vector system.

BACKGROUND OF THE INVENTION

Polyketides are a large, structurally diverse family of naturalproducts. Polyketides possess a broad range of biological activitiesincluding antibiotic and pharmacological properties. For example,polyketides are represented by such antibiotics as tetracyclines anderythromycin, anticancer agents including daunomycin,immunosuppressants, for example FK506 and rapamycin, and veterinaryproducts such as monensin and avermectin. Polyketides occur in mostgroups of organisms and are especially abundant in a class of mycelialbacteria, the actinomycetes, which produce various polyketides.

Polyketide synthases (PKSs) are multifunctional enzymes related to fattyacid synthases (FASs). PKSs catalyze the biosynthesis of polyketidesthrough repeated (decarboxylative) Claisen condensations betweenacylthioesters, usually acetyl, propionyl, malonyl or methylmalonyl.Following each condensation, they introduce structural variability intothe product by catalyzing all, part, or none of a reductive cyclecomprising a ketoreduction, dehydration, and enoylreduction on theβ-keto group of the growing polyketide chain. After the carbon chain hasgrown to a length characteristic of each specific product, it isreleased from the synthase by thiolysis or acyltransfer. Thus, PKSsconsist of families of enzymes which work together to produce a givenpolyketide. It is the controlled variation in chain length, choice ofchain-building units, and the reductive cycle, genetically programmedinto each PKS, that contributes to the variation seen among naturallyoccurring polyketides.

Two general classes of PKSs exist. One class, known as Type I PKSs, isrepresented by the PKSs for macrolides such as erythromycin. These“complex” or “modular” PKSs include assemblies of several largemultifunctional proteins carrying, between them, a set of separateactive sites for each step of carbon chain assembly and modification(Cortes, J. et al. Nature (1990) 348:176; Donadio, S. et al. Science(1991) 252:675; MacNeil, D. J. et al. Gene (1992) 115:119). Structuraldiversity occurs in this class from variations in the number and type ofactive sites in the PKSs. This class of PKSs displays a one-to-onecorrelation between the number and clustering of active sites in theprimary sequence of the PKS and the structure of the polyketidebackbone.

The second class of PKSs, called Type II PKSs, is represented by thesynthases for aromatic compounds. Type II PKSs have a single set ofiteratively used active sites (Bibb, M. J. et al. EMBO J. (1989) 8:2727;Sherman, D. H. et al. EMBO J. (1989) 8:2717; Fernandez-Moreno, M. A. etal. J. Biol. Chem. (1992) 267:19278).

Streptomyces is an actinomycete which is an abundant producer ofaromatic polyketides. In each Streptomyces aromatic PKS so far studied,carbon chain assembly requires the products of three open reading frames(ORFs). ORF1 encodes a ketosynthase (KS) and an acyltransferase (AT)active site; ORF2 encodes a protein similar to the ORF1 product butlacking the KS and AT motifs; and ORF3 encodes a discrete acyl carrierprotein (ACP).

Streptomyces coelicolor produces the blue-pigmented polyketide,actinorhodin. The actinorhodin gene cluster (act), has been cloned(Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462; Malpartida, F.and Hopwood, D. A. Mol. Gen. Genet. (1986) 205:66) and completelysequenced (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992)267:19278; Hallam, S. E. et al. Gene (1988) 74:305; Fernandez-Moreno, M.A. et al. Cell (1991) 66:769; Caballero, J. et al. Mol. Gen. Genet.(1991) 230:401). The cluster encodes the PKS enzymes described above, acyclase and a series of tailoring enzymes involved in subsequentmodification reactions leading to actinorhodin, as well as proteinsinvolved in export of the antibiotic and at least one protein thatspecifically activates transcription of the gene cluster. Other genesrequired for global regulation of antibiotic biosynthesis, as well asfor the supply of starter (acetyl CoA) and extender (malonyl CoA) unitsfor polyketide biosynthesis, are located elsewhere in the genome.

The act gene cluster from S. coelicolor has been used to produceactinorhodin in S. parvulus. Malpartida, F. and Hopwood, D. A. Nature(1984) 309:462. Bartel et al. J. Bacteriol. (1990) 172:4816-4826,recombinantly produced aloesaponarin II using S. galilaeus transformedwith an S. coelicolor act gene cluster consisting of four genetic loci,acti, actIII, actIV and actVII. Hybrid PKSS, including the basic actgene set but with ACP genes derived from granaticin, oxytetracycline,tetracenomycin and frenolicin PKSs, have also been designed which areable to express functional synthases. Khosla, C. et al. J. Bacteriol.(1993) 175:2197-2204. Hopwood, D. A. et al. Nature (1985) 314:642-644,describes the production of hybrid polyketides, using recombinanttechniques. Sherman, D. H. et al. J. Bacteriol. (1992) 174:6184-6190,reports the transformation of various S. coelicolor mutants, lackingdifferent components of the act PKS gene cluster, with the correspondinggranaticin (gra) genes from S. violaceoruber, in trans.

However, no one to date has described the recombinant production ofpolyketides using genetically engineered host cells which substantiallylack their entire native PKS gene clusters.

SUMMARY OF THE INVENTION

The present invention provides for novel polyketides and novel methodsof efficiently producing both new and known polyketides, usingrecombinant technology. In particular, a novel host-vector system isused to produce PKSs which in turn catalyze the production of a varietyof polyketides. Such polyketides are useful as antibiotics, antitumoragents, immunosuppressants and for a wide variety of otherpharmacological purposes.

Accordingly, in one embodiment, the invention is directed to agenetically engineered cell which expresses a polyketide synthase (PKS)gene cluster in its native, nontransformed state, the geneticallyengineered cell substantially lacking the entire native PKS genecluster.

In another embodiment, the invention is directed to the geneticallyengineered cell as described above, wherein the cell comprises:

(a) a replacement PKS gene cluster which encodes a PKS capable ofcatalyzing the synthesis of a polyketide; and

(b) one or more control sequences operatively linked to the PKS genecluster, whereby the genes in the gene cluster can be transcribed andtranslated in the genetically engineered cell,

with the proviso that when the replacement PKS gene cluster comprises anentire PKS gene set, at least one of the PKS genes or control elementsis heterologous to the cell.

In particularly preferred embodiments, the genetically engineered cellis Streptomyces coelicolor, the cell substantially lacks the entirenative actinorhodin PKS gene cluster and the replacement PKS genecluster comprises a first gene encoding a PKS ketosynthase and a PKSacyltransferase active site (KS/AT), a second gene encoding a PKS chainlength determining factor (CLF), and a third gene encoding a PKS acylcarrier protein (ACP).

In another embodiment, the invention is directed to a method forproducing a recombinant polyketide comprising:

(a) providing a population of cells as described above; and

(b) culturing the population of cells under conditions whereby thereplacement PKS gene cluster present in the cells, is expressed.

In still another embodiment, the invention is directed to a method forproducing a recombinant polyketide comprising:

a. inserting a first portion of a replacement PKS gene cluster into adonor plasmid and inserting a second portion of a replacement PKS genecluster into a recipient plasmid, wherein the first and second portionscollectively encode a complete replacement PKS gene cluster, and furtherwherein:

i. the donor plasmid expresses a gene which encodes a first selectionmarker and is capable of replication at a first, permissive temperatureand incapable of replication at a second, non-permissive temperature;

ii. the recipient plasmid expresses a gene which encodes a secondselection marker; and

iii. the donor plasmid comprises regions of DNA complementary to regionsof DNA in the recipient plasmid, such that homologous recombination canoccur between the first portion of the replacement PKS gene cluster andthe second portion of the replacement gene cluster, whereby a completereplacement gene cluster can be generated;

b. transforming the donor plasmid and the recipient plasmid into a hostcell and culturing the transformed host cell at the first, permissivetemperature and under conditions which allow the growth of host cellswhich express the first and/or the second selection markers, to generatea first population of cells;

c. culturing the first population of cells at the second, non-permissivetemperature and under conditions which allow the growth of cells whichexpress the first and/or the second selection markers, to generate asecond population of cells which includes host cells which contain arecombinant plasmid comprising a complete PKS replacement gene cluster;

d. transferring the recombinant plasmid from the second population ofcells into the genetically engineered cell of claim 1 to generatetransformed genetically engineered cells; and

e. culturing the transformed genetically engineered cells underconditions whereby the replacement PKS gene cluster present in the cellsis expressed.

In yet another embodiment, the invention is directed to a polyketidecompound having the structural formula (I)

wherein:

R¹ is selected from the group consisting of hydrogen and lower alkyl andR² is selected from the group consisting of hydrogen, lower alkyl andlower alkyl ester, or wherein R¹ and R² together form a lower alkylenebridge optionally substituted with one to four hydroxyl or lower alkylgroups;

R³ and R⁵ are independently selected from the group consisting ofhydrogen, halogen, lower alkyl, lower alkoxy, amino, lower alkyl mono-or di-substituted amino and nitro;

R⁴ is selected from the group consisting of halogen, lower alkyl, loweralkoxy, amino, lower alkyl mono- or di-substituted amino and nitro;

R⁶ is selected from the group consisting of hydrogen, lower alkyl, and—CHR⁷—(CO)R⁸ where R⁷ and R⁸ are independently selected from the groupconsisting of hydrogen and lower alkyl; and

i is 1, 2 or 3.

In another embodiment, the invention related to novel polyketides havingthe structures

In another embodiment, the invention is directed to a polyketidecompound formed by catalytic cyclization of an enzyme-bound ketidehaving the structure (II)

wherein:

R¹¹ is selected from the group consisting of methyl, —CH₂(CO)CH₃ and—CH₂(CO)CH₂(CO)CH₃;

R¹² is selected from the group consisting of —S—E and —CH₂(CO)—S—E,wherein E represents a polyketide synthase produced by the geneticallyengineered cells above; and

one of R¹³ and R¹⁴ is hydrogen and the other is hydroxyl, or R¹³ and R¹⁴together represent carbonyl.

In still another embodiment, the invention is directed to a method forproducing an aromatic polyketide, comprising effecting cyclization of anenzyme-bound ketide having the structure (II), wherein cyclization isinduced by the polyketide synthase.

In a further embodiment, the invention is directed to a polyketidecompound having the structural formula (III)

wherein R² and R⁴ are as defined above and i is 0, 1 or 2.

In another embodiment, the invention is directed to a polyketidecompound having the structural formula (IV)

wherein R², R⁴ and i are as defined above for structural formula (III).

In still anther embodiment, the invention is directed to a polyketidecompound having the structural formula (V)

wherein R², R⁴ and i are as defined above for structural formula (III).

These and other embodiments of the subject invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the gene clusters for act, gra, and tcm PKSs and cyclases.

FIG. 2 shows the strategy for making S. coelicolor CH999. FIG. 2Adepicts the structure of the act gene cluster present on the S.coelicolor CH1 chromosome. FIG. 2B shows the structure of pLRemEts andFIG. 2C shows the portion of the CH999 chromosome with the act genecluster deleted.

FIG. 3 is a diagram of plasmid pRM5.

FIG. 4 schematically illustrates formation of aloesaponarin II (2) andits carboxylated analog,3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid (1) as describedin Example 3.

FIG. 5 provides the structures of actinorhodin (3), granaticin (4),tetracenomycin (5) and mutactin (6), referenced in Example 4.

FIG. 6 schematically illustrates the preparation, via cyclization of thepolyketide precursors, of aloesaponarin II (2), its carboxylated analog,3,8-dihydroxy-1-methylanthraquinone-2-carboxylic acid (1),tetracenomycin (5) and new compound RM20 (9), as explained in Example 4,part (A).

FIG. 7 schematically illustrates the preparation, via cyclization of thepolyketide precursors, of frenolicin (7), nanomycin (8) and actinorhodin(3).

FIGS. 88A-8C schematically illustrates the preparation, via cyclizationof the polyketide precursors, of novel compounds RM20 (9), RM18 (10),RM18b (11), SEK4 (12), SEK15 (13), RM20b (14), RM20c (15) and SEK15b(16).

FIG. 9 depicts the genetic model for the 6-deoxyerythronolide B synthase(DEBS).

FIG. 10 shows the strategy for the construction of recombinant modularPKSs.

FIG. 11 is a diagram of plasmid pCK7.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, microbiology, molecularbiology and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Transcription and Translation (B. Hames & S. Higgins, eds., CurrentEdition).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, reference to “a polyketide” includesmixtures of polyketides, reference to “a polyketide synthase” includesmixtures of polyketide synthases, and the like.

A. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “replacement PKS gene cluster” is meant any set of PKS genes capableof producing a functional PKS when under the direction of one or morecompatible control elements, as defined below, in a host celltransformed therewith. A functional PKS is one which catalyzes thesynthesis of a polyketide. The term “replacement PKS gene cluster”encompasses one or more genes encoding for the various proteinsnecessary to catalyze the production of a polyketide. A “replacement PKSgene cluster” need not include all of the genes found in thecorresponding cluster in nature. Rather, the gene cluster need onlyencode the necessary PKS components to catalyze the production of anactive polyketide. Thus, as explained further below, if the gene clusterincludes, for example, eight genes in its native state and only three ofthese genes are necessary to provide an active polyketide, only thesethree genes need be present. Furthermore, the cluster can include PKSgenes derived from a single species, or may be hybrid in nature with,e.g., a gene derived from a cluster for the synthesis of a particularpolyketide replaced with a corresponding gene from a cluster for thesynthesis of another polyketide. Hybrid clusters can include genesderived from both Type I and Type II PKSs. As explained above, Type IPKSs include several large multifunctional proteins carrying, betweenthem, a set of separate active sites for each step of carbon chainassembly and modification. Type II PKSs, on the other hand, have asingle set of iteratively used active sites. These classifications arewell known. See, e.g., Hopwood, D. A. and Khosla, C. Secondarymetabolites: their function and evolution (1992) Wiley Chichester (CibaFoundation Symposium 171) p 88-112; Bibb, M. J. et al. EMBO J. (1989)8:2727; Sherman, D. H. et al. EMBO J. (1989) 8:2717; Fernandez-Moreno,M. A. et al. J. Biol. Chem. (1992) 267:19278); Cortes, J. et al. Nature(1990) 348:176; Donadio, S. et al. Science (1991) 252:675; MacNeil, D.J. et al. Gene (1992) 115:119. Hybrid clusters are exemplified hereinand are described further below. The genes included in the gene clusterneed not be the native genes, but can be mutants or analogs thereof.Mutants or analogs may be prepared by the deletion, insertion orsubstitution of one or more nucleotides of the coding sequence.Techniques for modifying nucleotide sequences, such as site-directedmutagenesis, are described in, e.g., Sambrook et al., supra; DNACloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

A “replacement PKS gene cluster” may also contain genes coding formodifications to the core polyketide catalyzed by the PKS, including,for example, genes encoding hydroxylases, methylases or other alkylases,oxidases, reductases, glycotransferases, lyases, ester or amidesynthases, and various hydrolases such as esterases and amidases.

As explained further below, the genes included in the replacement genecluster need not be on the same plasmid or if present on the sameplasmid, can be controlled by the same or different control sequences.

By “genetically engineered host cell” is meant a host cell where thenative PKS gene cluster has been deleted using recombinant DNAtechniques. Thus, the term would not encompass mutational eventsoccurring in nature. A “host cell” is a cell derived from a procaryoticmicroorganism or a eucaryotic cell line cultured as a unicellularentity, which can be, or has been, used as a recipient for recombinantvectors bearing the PKS gene clusters of the invention. The termincludes the progeny of the original cell which has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell which are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a desired PKS, are includedin the definition, and are covered by the above terms.

The term “heterologous” as it relates to nucleic acid sequences such ascoding sequences and control sequences, denotes sequences that are notnormally associated with a region of a recombinant construct, and/or arenot normally associated with a particular cell. Thus, a “heterologous”region of a nucleic acid construct is an identifiable segment of nucleicacid within or attached to another nucleic acid molecule that is notfound in association with the other molecule in nature. For example, aheterologous region of a construct could include a coding sequenceflanked by sequences not found in association with the coding sequencein nature. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,synthetic sequences having codons different from the native gene).Similarly, a host cell transformed with a construct which is notnormally present in the host cell would be considered heterologous forpurposes of this invention. Allelic variation or naturally occurringmutational events do not give rise to heterologous DNA, as used herein.

A “coding sequence” or a sequence which “encodes” a particular PKS, is anucleic acid sequence which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom procaryotic or eucaryotic MRNA, genomic DNA sequences fromprocaryotic or eucaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence will usually be located 3′ to thecoding sequence.

A “nucleic acid” sequence can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. A transcription termination sequence will usually belocated 3′ to the coding sequence.

DNA “control sequences” refers collectively to promoter sequences,ribosome binding sites, polyadenylation signals, transcriptiontermination sequences, upstream regulatory domains, enhancers, and thelike, which collectively provide for the transcription and translationof a coding sequence in a host cell. Not all of these control sequencesneed always be present in a recombinant vector so long as the desiredgene is capable of being transcribed and translated.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent-between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

By “selection marker” is meant any genetic marker which can be used toselect a population of cells which carry the marker in their genome.Examples of selection markers include: auxotrophic markers by whichcells are selected by their ability to grow on minimal media with orwithout a nutrient or supplement, e.g., thymidine, diaminopimelic acidor biotin; metabolic markers by which cells are selected for theirability to grow on minimal media containing the appropriate sugar as thesole carbon source or the ability of cells to form colored coloniescontaining the appropriate dyes or chromogenic substrates; and drugresistance markers by which cells are selected by their ability to growon media containing one or more of the appropriate drugs, e.g.,tetracycline, ampicillin, kanamycin, streptomycin or nalidixic acid.

“Recombination” is a the reassortment of sections of DNA sequencesbetween two DNA molecules. “Homologous recombination” occurs between twoDNA molecules which hybridize by virtue of homologous or complementarynucleotide sequences present in each DNA molecule.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl,tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred alkylgroups herein contain 1 to 12 carbon atoms. The term “lower alkyl”intends an alkyl group of one to six carbon atoms, preferably one tofour carbon atoms.

The term “alkylene” as used herein refers to a difunctional saturatedbranched or unbranched hydrocarbon chain containing from 1 to 24 carbonatoms, and includes, for example, methylene (—CH₂—), ethylene(—CH₂—CH₂—), propylene (—CH₂—CH₂—CH₂—), 2-methylpropylene[—CH₂—CH(CH₃)—CH₂—], hexylene [—(CH₂)₆—] and the like. “Lower alkylene”refers to an alkylene group of 1 to 6, more preferably 1 to 4, carbonatoms.

The term “alkoxy” as used herein intends an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group may bedefined as —OR where R is alkyl as defined above. A “lower alkoxy” groupintends an alkoxy group containing one to six, more preferably one tofour, carbon atoms.

“Halo” or “halogen” refers to fluoro, chloro, bromo or iodo, and usuallyrelates to halo substitution for a hydrogen atom in an organic compound.Of the halos, chloro and fluoro are generally preferred.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally substituted alkylene”means that an alkylene moiety may or may not be substituted and that thedescription includes both unsubstituted alkylene and alkylene wherethere is substitution.

B. General Methods

Central to the present invention is the discovery of a host-vectorsystem for the efficient recombinant production of both novel and knownpolyketides. In particular, the invention makes use of geneticallyengineered cells which have their naturally occurring PKS genessubstantially deleted. These host cells can be transformed withrecombinant vectors, encoding a variety of PKS gene clusters, for theproduction of active polyketides. The invention provides for theproduction of significant quantities of product at an appropriate stageof the growth cycle. The polyketides so produced can be used astherapeutic agents, to treat a number of disorders, depending on thetype of polyketide in question. For example, several of the polyketidesproduced by the present method will find use as immunosuppressants, asanti-tumor agents, as well as for the treatment of viral, bacterial andparasitic infections. The ability to recombinantly produce polyketidesalso provides a powerful tool for characterizing PKSs and the mechanismof their actions.

More particularly, host cells for the recombinant production of thesubject polyketides can be derived from any organism with the capabilityof harboring a recombinant PKS gene cluster. Thus, the host cells of thepresent invention can be derived from either procaryotic or eucaryoticorganisms. However, preferred host cells are those constructed from theactinomycetes, a class of mycelial bacteria which are abundant producersof a number of polyketides. A particularly preferred genus for use withthe present system is Streptomyces. Thus, for example, S. ambofaciens,S. avermitilis, S. azureus, S. cinnamonensis, S. coelicolor, S. curacoi,S. erythraeus, S. fradiae, S. galilaeus, S. glaucescens, S.hygroscopicus, S. lividans, S. parvulus, S. peucetius, S. rimosus, S.roseofulvus, S. thermotolerans, S. violaceoruber, among others, willprovide convenient host cells for the subject invention, with S.coelicolor being preferred. (See, e.g., Hopwood, D. A. and Sherman, D.H. Ann. Rev. Genet. (1990) 24:37-66; O'Hagan, D. The PolyketideMetabolites (Ellis Horwood Limited, 1991), for a description of variouspolyketide-producing organisms and their natural products.)

The above-described cells are genetically engineered by deleting thenaturally occurring PKS genes therefrom, using standard techniques, suchas by homologous recombination. (See, e.g., Khosla, C. et al. Molec.Microbiol. (1992) 6:3237). Exemplified herein is a geneticallyengineered S. coelicolor host cell. Native strains of S. coelicolorproduce a PKS which catalyzes the biosynthesis of the aromaticpolyketide actinorhodin (structure 3, FIG. 5). The novel strain, S.coelicolor CH999 (FIG. 2C and described in the examples), wasconstructed by deleting, via homologous recombination, the entirenatural act cluster from the chromosome of S. coelicolor CH1 (Khosla, C.Molec. Microbiol. (1992) 6:3237), a strain lacking endogenous plasmidsand carrying a stable mutation that blocks biosynthesis of anotherpigmented S. coelicolor antibiotic, undecylprodigiosin.

The host cells described above can be transformed with one or morevectors, collectively encoding a functional PKS set. The vector(s) caninclude native or hybrid combinations of PKS subunits, or mutantsthereof. As explained above, the replacement gene cluster need notcorrespond to the complete native gene cluster but need only encode thenecessary PKS components to catalyze the production of a polyketide. Forexample, in each Streptomyces aromatic PKS so far studied, carbon chainassembly requires the products of three open reading frames (ORFs). ORF1encodes a ketosynthase (KS) and an acyltransferase (AT) active site(KS/AT); as elucidated herein, ORF2 encodes a chain length determiningfactor (CLF), a protein similar to the ORF1 product but lacking the KSand AT motifs; and ORF3 encodes a discrete acyl carrier protein (ACP).Some gene clusters also code for a ketoreductase (KR) and a cyclase,involved in cyclization of the nascent polyketide backbone. (See FIG. 1for a schematic representation of three PKS gene clusters.) However, ithas been found that only the KS/AT, CLF, and ACP, need be present inorder to produce an identifiable polyketide. Thus, in the case ofaromatic PKSs derived from Streptomyces, these three genes, without theother components of the native clusters, can be included in one or morerecombinant vectors, to constitute a “minimal” replacement PKS genecluster.

Furthermore, the recombinant vector(s) can include genes from a singlePKS gene cluster, or may comprise hybrid replacement PKS gene clusterswith, e.g., a gene for one cluster replaced by the corresponding genefrom another gene cluster. For example, it has been found that ACPs arereadily interchangeable among different synthases without an effect onproduct structure. Furthermore, a given KR can recognize and reducepolyketide chains of different chain lengths. Accordingly, these genesare freely interchangeable in the constructs described herein. Thus, thereplacement clusters of the present invention can be derived from anycombination of PKS gene sets which ultimately function to produce anidentifiable polyketide.

Examples of hybrid replacement clusters include clusters with genesderived from two or more of the act gene cluster, frenolicin (fren),granaticin (gra), tetracenomycin (tcm), 6-methylsalicylic acid (6-msas),oxytetracycline (otc), tetracycline (tet), erythromycin (ery),griseusin, nanaomycin, medermycin, daunorubicin, tylosin, carbomycin,spiramycin, avermectin, monensin, nonactin, curamycin, rifamycin andcandicidin synthase gene clusters, among others. (For a discussion ofvarious PKSs, see, e.g., Hopwood, D. A. and Sherman, D. H. Ann. Rev.Genet. (1990) 24:37-66; O'Hagan, D. The Polyketide Metabolites (EllisHorwood Limited, 1991.)

More particularly, a number of hybrid gene clusters have beenconstructed herein, having components derived from the act, fren, tcmand gra gene clusters, as depicted in Tables 1 and 2. Several of thehybrid clusters were able to functionally express both novel and knownpolyketides in S. coelicolor CH999 (described above). However, otherhybrid gene clusters, as described above, can easily be produced andscreened using the disclosure herein, for the production of identifiablepolyketides. For example, a library of randomly cloned ORF1 and 2homologs, from a collection of actinomycetes, could be constructed andscreened for identifiable polyketides. Longer polyketides might also becyclized by replacing, e.g., an act, gra, fren or tcm cyclase gene witha homolog from a PKS gene cluster which produces a chain of the correctlength. Finally, a considerable degree of variability exists fornon-acetate starter units among certain naturally occurring aromaticPKSs; thus, these units can also be used for obtaining novel polyketidesvia genetic engineering.

Additionally, the recombinant vectors can include genes from a modularPKS gene cluster. Such gene clusters are described in further detailbelow.

The recombinant vectors, harboring the gene clusters described above,can be conveniently generated using techniques known in the art. Forexample, the PKS subunits of interest can be obtained from an organismthat expresses the same, using recombinant methods, such as by screeningcDNA or genomic libraries, derived from cells expressing the gene, or byderiving the gene from a vector known to include the same. The gene canthen be isolated and combined with other desired PKS subunits, usingstandard techniques. If the gene in question is already present in asuitable expression vector, it can be combined in situ, with, e.g.,other PKS subunits, as desired. The gene of interest can also beproduced synthetically, rather than cloned. The nucleotide sequence canbe designed with the appropriate codons for the particular amino acidsequence desired. In general, one will select preferred codons for theintended host in which the sequence will be expressed. The completesequence is assembled from overlapping oligonucleotides prepared bystandard methods and assembled into a complete coding sequence. See,e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311.

Mutations can be made to the native PKS subunit sequences and suchmutants used in place of the native sequence, so long as the mutants areable to function with other PKS subunits to collectively catalyze thesynthesis of an identifiable polyketide. Such mutations can be made tothe native sequences using conventional techniques such as by preparingsynthetic oligonucleotides including the mutations and inserting themutated sequence into the gene encoding a PKS subunit using restrictionendonuclease digestion. (See, e.g., Kunkel, T. A. Proc. Natl. Acad. Sci.USA (1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.)Alternatively, the mutations can be effected using a mismatched primer(generally 10-20 nucleotides in length) which hybridizes to the nativenucleotide sequence (generally cDNA corresponding to the RNA sequence),at a temperature below the melting temperature of the mismatched duplex.The primer can be made specific by keeping primer length and basecomposition within relatively narrow limits and by keeping the mutantbase centrally located. Zoller and Smith, Methods Enzymol. (1983)100:468. Primer extension is effected using DNA polymerase, the productcloned and clones containing the mutated DNA, derived by segregation ofthe primer extended strand, selected. Selection can be accomplishedusing the mutant primer as a hybridization probe. The technique is alsoapplicable for generating multiple point mutations. See, e.g.,Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409. PCRmutagenesis will also find use for effecting the desired mutations.

The gene sequences which collectively encode a replacement PKS genecluster, can be inserted into one or more expression vectors, usingmethods known to those of skill in the art. Expression vectors willinclude control sequences operably linked to the desired PKS codingsequence. Suitable expression systems for use with the present inventioninclude systems which function in eucaryotic and procaryotic host cells.However, as explained above, procaryotic systems are preferred, and inparticular, systems compatible with Streptomyces spp. are of particularinterest. Control elements for use in such systems include promoters,optionally containing operator sequences, and ribosome binding sites.Particularly useful promoters include control sequences derived from PKSgene clusters, such as one or more act promoters. However, otherbacterial promoters, such as those derived from sugar metabolizingenzymes, such as galactose, lactose (lac) and maltose, will also finduse in the present constructs. Additional examples include promotersequences derived from biosynthetic enzymes such as tryptophan (trp),the β-lactamase (bla) promoter system, bacteriophage lambda PL, and T5.In addition, synthetic promoters, such as the tac promoter (U.S. Pat.No. 4,551,433), which do not occur in nature also function in bacterialhost cells.

Other regulatory sequences may also be desirable which allow forregulation of expression of the PKS replacement sequences relative tothe growth of the host cell. Regulatory sequences are known to those ofskill in the art, and examples include those which cause the expressionof a gene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Other typesof regulatory elements may also be present in the vector, for example,enhancer sequences.

Selectable markers can also be included in the recombinant expressionvectors. A variety of markers are known which are useful in selectingfor transformed cell lines and generally comprise a gene whoseexpression confers a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium. Such markersinclude, for example, genes which confer antibiotic resistance orsensitivity to the plasmid. Alternatively, several polyketides arenaturally colored and this characteristic provides a built-in marker forselecting cells successfully transformed by the present constructs.

The various PKS subunits of interest can be cloned into one or morerecombinant vectors as individual cassettes, with separate controlelements, or under the control of, e.g., a single promoter. The PKSsubunits can include flanking restriction sites to allow for the easydeletion and insertion of other PKS subunits so that hybrid PKSs can begenerated. The design of such unique restriction sites is known to thoseof skill in the art and can be accomplished using the techniquesdescribed above, such as site-directed mutagenesis and PCR.

Using these techniques, a novel plasmid, PRM5, (FIG. 3 and Example 2)was constructed as a shuttle vector for the production of thepolyketides described herein. Plasmid pRM5 includes the act genesencoding the KS/AT (ORF1), CLF (ORF2) and ACP (ORF3) PKS subunits,flanked by PacI, NsiI and XbaI restriction sites. Thus, analogous PKSsubunits, encoded by other PKS genes, can be easily substituted for theexisting act genes. (See, e.g., Example 4, describing the constructionof hybrid vectors using pRM5 as the parent plasmid). The shuttle plasmidalso contains the act KR gene (actIII), the cyclase gene (actVII), and aputative dehydratase gene (actIV), as well as a ColEI replicon (to allowtransformation of E. coli), an appropriately truncated SCP2* (low copynumber) Streptomyces replicon, and the actII-ORF4 activator gene fromthe act cluster, which induces transcription from act promoters duringthe transition from growth phase to stationary phase in the vegetativemycelium. pRM5 carries the divergent actI/actIII promoter pair.

Methods for introducing the recombinant vectors of the present inventioninto suitable hosts are known to those of skill in the art and typicallyinclude the use of CaCl₂ or other agents, such as divalent cations andDMSO. DNA can also be introduced into bacterial cells byelectroporation. Once the PKSs are expressed, the polyketide producingcolonies can be identified and isolated using known techniques. Theproduced polyketides can then be further characterized.

As explained above, the above-described recombinant methods also findutility in the catalytic biosynthesis of polyketides by large, modularPKSs. For example, 6-deoxyerythronolide B synthase (DEBS) catalyzes thebiosynthesis of the erythromycin aglycone, 6-deoxyerythronolide B (17).Three open reading frames (eryAI, eryAII, and eryAIII) encode the DEBSpolypeptides and span 32 kb in the ery gene cluster of theSaccharopolyspora erythraea genome. The genes are organized in sixrepeated units, each designated a “module.” Each module encodes a set ofactive sites that, during polyketide biosynthesis, catalyzes thecondensation of an additional monomer onto the growing chain. Eachmodule includes an acyltransferase (AT), β-ketoacyl carrier proteinsynthase (KS), and acyl carrier protein (ACP) as well as a subset ofreductive active sites (β-ketoreductase (KR), dehydratase (DH), enoylreductase (ER)) (FIG. 9). The number of reductive sites within a modulecorresponds to the extent of β-keto reduction in each condensationcycle. The thioesterase (TE) encoded at the end of module appears tocatalyze lactone formation.

Due to the large sizes of eryAI, eryAII, and eryAIII, and the presenceof multiple active sites, these genes can be conveniently cloned into aplasmid suitable for expression in a genetically engineered host cell,such as CH999, using an in vivo recombination technique This technique,described in Example 5 and summarized in FIG. 10, utilizes derivativesof the plasmid pMAK705 (Hamilton et al. (1989) J. Bacteriol. 171:4617)to permit in vivo recombination between a temperature-sensitive donorplasmid, which is capable of replication at a first, permissivetemperature and incapable of replication at a second, non-permissivetemperature, and recipient plasmid. The eryA genes thus cloned gavepCK7, a derivative of pRM5 (McDaniel et al. (1993) Science 262:1546). Acontrol plasmid, pCK7f, was constructed to carry a frameshift mutationin eryAI. pCK7 and pCK7f possess a ColEI replicon for geneticmanipulation in E. coli as well as a truncated SCP2* (low copy number)Streptomyces replicon. These plasmids also contain the divergentactI/actIII promoter pair and actII-ORF4, an activator gene, which isrequired for transcription from these promoters and activates expressionduring the transition from growth to stationary phase in the vegetativemycelium. High-level expression of PKS genes occurs at the onset ofstationary phase of mycelial growth; the recombinant strains thereforeproduce “reporter” polyketides as secondary metabolites in aquasi-natural manner.

The method described above for producing polyketides synthesized bylarge, modular PKSs may be used to produce other polyketides assecondary metabolites such as sugars, β-lactams, fatty acids,aminoglycosides, terpinoids, non-ribosomal peptides, prostanoid hormonesand the like.

Using the above recombinant methods, a number of polyketides have beenproduced. These compounds have the general structure (I)

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and i are as defined above. Onegroup of such compounds are wherein: R¹ is lower alkyl, preferablymethyl; R², R³ and R⁶ are hydrogen; R⁶ is —CHR⁷—(Co)—R⁸; and i is 0. Asecond group of such compounds are wherein: R¹ and R⁶ are lower alkyl,preferably methyl; R², R³ and R⁵ are hydrogen; and i is 0. Still a thirdgroup of such compounds are wherein: R¹ and R² are linked together toform a lower alkylene bridge —CHR⁹—CHR¹⁰ wherein R⁹ and R¹⁰ areindependently selected from the group consisting of hydrogen, hydroxyland lower alkyl, e.g., —CH₂—CHOH—; R³ and R⁵ are hydrogen; R⁶ is—CHR⁷—(Co)—R⁸ where R⁸ is hydrogen or lower alkyl, e.g., —CH₂—(CO)—CH₃;and i is 0. Specific such compounds include the following compounds 9,10 and 11 as follows:

Other novel polyketides within the scope of the invention are thosehaving the structure

Preparation of compounds 9, 10, 11, 12, 13, 14, 15 and 16 is effected bycyclization of an enzyme-bound polyketide having the structure (II)

wherein R¹¹, R¹², R¹³ and R¹⁴ and E are as defined earlier herein.Examples of such compounds include: a first group wherein R¹¹ is methyland R¹² is —CH₂(CO)—S—E; a second group wherein R¹¹ is —CH₂(CO)CH₃ andR¹² is —S—E; a third group wherein R¹¹ is —CH₂(CO)CH₃ and R¹² isCH₂(CO)—S—E; and a fourth group wherein R¹¹ is —CH₂(CO)CH₂(CO)CH₃ andR¹² is —CH₂(CO)—S—E (see FIG. 8 for structural exemplification).

The remaining structures encompassed by generic formula (I)—i.e.,structures other than 9, 10 and 11—may be prepared from structures 9, 10or 11 using routine synthetic organic methods well-known to thoseskilled in the art of organic chemistry, e.g., as described by H.O.House, Modern Synthetic Reactions, Second Edition (Menlo Park, Calif.:The Benjamin/Cummings Publishing Company, 1972), or by J. March,Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992), the disclosures of which arehereby incorporated by reference. Typically, as will be appreciated bythose skilled in the art, incorporation of substituents on the aromaticrings will involve simple electrophilic aromatic addition reactions.Structures 12 and 13 may be modified in a similar manner to producepolyketides which are also intended to be within the scope of thepresent invention.

In addition, the above recombinant methods have been used to producepolyketide compound having the general structure (III)

general structure (IV)

and general structure (V)

Particularly preferred compounds of structural formulas (III), (IV) and(V) are wherein: R² is hydrogen and i is 0.

C. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Materials and Methods

Bacterial strains, plasmids, and culture conditions. S. coelicolor CH999was used as a host for transformation by all plasmids. The constructionof this strain is described below. DNA manipulations were performed inEscherichia coli MC1061. Plasmids were passaged through E. coli ET12567(dam dcm hsds Cm^(r)) (MacNeil, D. J. J. Bacteriol. (1988) 170:5607) togenerate unmethylated DNA prior to transformation of S. coelicolor. E.coli strains were grown under standard conditions. S. coelicolor strainswere grown on R2YE agar plates (Hopwood, D. A. et al. Geneticmanipulation of Streptomyces. A laboratory manual. The John InnesFoundation: Norwich, 1985).

Manipulation of DNA and organisms. Polymerase chain reaction (PCR) wasperformed using Taq polymerase (Perkin Elmer Cetus) under conditionsrecommended by the enzyme manufacturer. Standard in vitro techniqueswere used for DNA manipulations (Sambrook, et al. Molecular Cloning: ALaboratory Manual (Current Edition)). E. coli was transformed with aBio-Rad E. Coli Pulsing apparatus using protocols provided by Bio-Rad.S. coelicolor was transformed by standard procedures (Hopwood, D. A. etal. Genetic manipulation of Streptomyces. A laboratory manual. The JohnInnes Foundation: Norwich, 1985) and transformants were selected using 2ml of a 500 mg/ml thiostrepton overlay.

Construction of plasmids containing recombinant PKSs. All plasmids arederivatives of pRM5, described below. fren PKS genes were amplified viaPCR with 5′ and 3′ restriction sites flanking the genes in accordancewith the location of cloning sites on pRM5 (i.e. PacI-NsiI for ORF1,NsiI-XbaI for ORF2, and XbaI-PstI for ORF3). Following subcloning andsequencing, the amplified fragments were cloned in place of thecorresponding fragments in pRM5 to generate the plasmids fortransformation.

Production and purification of polyketides. For initial screening, allstrains were grown at 30° C. as confluent lawns on 10-30 plates eachcontaining approximately 30 ml of agar medium for 6-8 days. Additionalplates were made as needed to obtain sufficient material for completecharacterization. CH999 was a negative control when screening forpotential polyketides. The agar was finely chopped and extracted withethyl acetate/1% acetic acid or ethyl acetate:methanol (4:1)/1% aceticacid. The concentrated extract was then flashed through a silica gel(Baker 40 mm) chromatography column in ethyl acetate/1% acetic acid.Alternatively, the extract was applied to a Florisil column (FisherScientific) and eluted with ethyl acetate:ethanol:acetic acid (17:2:1).The primary yellow fraction was further purified via high-performanceliquid chromatography (HPLC) using a 20-60% acetonitrile/water/1% aceticacid gradient on a preparative reverse phase (C-18) column (Beckman).Absorbance was monitored at 280 nm and 410 nm. In general, the yield ofpurified product from these strains was approximately 10 mg/l forcompounds 1 and 2 (FIG. 4), and 5 mg/l for compounds 7 and 8 (FIG. 7).

SEK4, (12), was produced and purified as follows. CH999/pSEK4 was grownon 90 agar plates (˜34 ml/plate) at 30° C. for 7 days. The agar waschopped and extracted with ethyl acetate/methanol (4/1) in the presenceof 1% acetic acid (3×1000 ml). Following removal of the solvent undervacuum, 200 ml of ethyl acetate containing 1% acetic acid were added.The precipitate was filtered and discarded, and the solvent wasevaporated to dryness. The product mixture was applied to a Florisilcolumn (Fisher Scientific), and eluted with ethyl acetate containing 3%acetic acid. The first 100 ml fraction was collected, and concentrateddown to 5 ml. 1 ml methanol was added, and the mixture was kept at 4° C.overnight. The precipitate was collected by filtration, and washed withethyl acetate to give 850 mg of pure product. R_(f)=0.48 (ethyl acetatewith 1% acetic acid). Results from NMR spectroscopy on SEK4 are reportedin Table 4. FAB HRMS (NBA), M+^(H)+, calculated m/e 319.0818, observedm/e 319.0820.

To produce SEK15 (13) and SEK15b (16), CH999/pSEK15 was grown on 90 agarplates, and the product was extracted in the same manner as SEK4. Themixture was applied to a Florisil column (ethyl acetate with 5% aceticacid), and fractions containing the major products were combined andevaporated to dryness. The products were further purified usingpreparative C-18 reverse phase HPLC (Beckman) (mobile phase:acetonitrile/water=1/10 to 3/5 gradient in the presence of 1% aceticacid). The yield of SEK15, (13), was 250 mg. R_(f)=0.41 (ethyl acetatewith 1% acetic acid). Results from NMR spectroscopy on SEK4 are reportedin Table 4. FAB HRMS (NBA), M+H+, calculated m/e 385.0923, observed m/e385.0920.

[1,2-¹³C₂] acetate feeding experiments. Two 2 l flasks each containing400ml of modified NMP medium (Strauch, E. et al. Mol. Microbiol. (1991)5:289) were inoculated with spores of S. coelicolor CH999/pRM18,CH999/pSEK4 or CH999/pSEK15, and incubated in a shaker at 30 degrees C.and 300 rpm. To each flask, 50 mg of sodium [1,2-¹³C₂] acetate (Aldrich)was added at 72 and 96 hrs. After 120 hrs, the cultures were pooled andextracted with two 500 ml volumes of ethyl acetate/1% acetic acid. Theorganic phase was kept and purification proceeded as described above.¹³C NMR data indicate approximately a 2-3% enrichment for theCH999/pRM18 product; a 0.5-1% enrichment for SEK4 and a 1-2% enrichmentfor SEK15.

NMR Spectroscopy. All spectra were recorded on a Varian XL-400 exceptfor HETCOR analysis of RM18 (10) (FIG. 8), which was performed on aNicolet NT-360. ¹³C spectra were acquired with continuous broadbandproton decoupling. For NOE studies of RM18 (10), the one-dimensionaldifference method was employed. All compounds were dissolved in DMSO-d₆(Sigma, 99+ atom % D) and spectra were referenced internally to thesolvent. Hydroxyl resonances were identified by adding D₂O (Aldrich, 99atom % D) and checking for disappearance of signal.

EXAMPLE 1 Production of S. coelicolor CH999

An S. coelicolor host cell, genetically engineered to remove the nativeact gene cluster, and termed CH999, was constructed using S. coelicolorCH1 (Khosla, C. Molec. Microbiol. (1992) 6:3237), using the strategydepicted in FIG. 2. (CH1 is derived from S. coelicolor B385 (Rudd,B.A.M. Genetics of Pigmented Secondary Metabolites in Streptomycescoelicolor (1978) Ph.D. Thesis, University of East Anglia, Norwich,England.) CH1 includes the act gene cluster which codes for enzymesinvolved in the biosynthesis and export of the polyketide antibioticactinorhodin. The cluster is made up of the PKS genes, flanked byseveral post-PKS biosynthetic genes including those involved incyclization, aromatization, and subsequent chemical tailoring (FIG. 2A).Also present are the genes responsible for transcriptional activation ofthe act genes. The act gene cluster was deleted from CH1 usinghomologous recombination as described in Khosla, C. et al. Molec.Microbiol. (1992) 6:3237.

In particular, plasmid pLRermEts (FIG. 2B) was constructed with thefollowing features: a ColEI replicon from pBR322, the temperaturesensitive replicon from pSG5 (Muth, G. et al. Mol. Gen. Genet. (1989)219:341), ampicillin and thiostrepton resistance markers, and adisruption cassette including a 2 kb BamHI/XhoI fragment from the 5′ endof the act cluster, a 1.5 kb ermE fragment (Khosla, C. et al. Molec.Microbiol. (1992) 6:3237), and a 1.9 kb SphI/PstI fragment from the 3′end of the act cluster. The 5′ fragment extended from the BamHI site 1(Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462; Malpartida, F.and Hopwood, D. A. Mol. Gen. Genet. (1986) 205:66) downstream to a XhoIsite. The 3′ fragment extended from PstI site 20 upstream to SphI site19.2 (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278).The 5′ and 3′ fragments (shown as hatched DNA in FIG. 2) were cloned inthe same relative orientation as in the act cluster. CH1 was transformedwith pLRermEts. The plasmid was subsequently cured from candidatetransformants by streaking non-selectively at 39° C. Several coloniesthat were lincomycin resistant, thiostrepton sensitive, and unable toproduce actinorhodin, were isolated and checked via Southern blotting.One of them was designated CH999.

EXAMPLE 2 Production of the Recombinant Vector pRM5

pRM5 (FIG. 3) was the shuttle plasmid used for expressing PKSs in CH999.It includes a ColEI replicon to allow genetic engineering in E. coli, anappropriately truncated SCP2* (low copy number) Streptomyces replicon,and the actII-ORF4 activator gene from the act cluster, which inducestranscription from act promoters during the transition from growth phaseto stationary phase in the vegetative mycelium. As shown in FIG. 3, pRM5carries the divergent actI/actIII promoter pair, together withconvenient cloning sites to facilitate the insertion of a variety ofengineered PKS genes downstream of both promoters. pRM5 lacks the parlocus of SCP2*; as a result the plasmid is slightly unstable (approx. 2%loss in the absence of thiostrepton). This feature was deliberatelyintroduced in order to allow for rapid confirmation that a phenotype ofinterest could be unambiguously assigned to the plasmid-borne mutantPKS. The recombinant PKSs from pRM5 are expressed approximately at thetransition from exponential to stationary phase of growth, in goodyields.

pRM5 was constructed as follows. A 10.5 kb SphI/HindIII fragment frompIJ903 (containing a portion of the fertility locus and the origin ofreplication of SCP2 as well as the colEI origin of replication and theβ-lactamase gene from pBR327) (Lydiate, D. J. Gene (1985) 35:223) wasligated with a 1.5 kb,HindIII/SphI tsr gene cassette to yield pRM1. pRM5was constructed by inserting the following two fragments between theunique HindIII and EcoRI sites of pRM1: a 0.3 kb HindIII/HpaI (blunt)fragment carrying a transcription terminator from phage fd (Khosla, C.et al. Molec. Microbiol. (1992) 6:3237), and a 10 kb fragment from theact cluster extending from the NcoI site (1 kb upstream of theactII-ORF4 activator gene) (Hallam, S. E. et al. Gene (1988) 74:305;Fernandez-Moreno, M. A. et al. Cell (1991) 66:769; Caballero, J. L. Mol.Gen. Genet. (1991) 230:401) to the PstI site downstream of theactI-VII-IV genes (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992)267:19278).

To facilitate the expression of any desired recombinant PKS under thecontrol of the actI promoter (which is activated by the actII-ORF4 geneproduct), restriction sites for PacI, NsiI, XbaI, and PstI wereengineered into the act DNA in intercistronic positions. In pRM5, aswell as in all other PKS expression plasmids described here, ORF1, 2,and 3 alleles were cloned between these sites as cassettes engineeredwith their own RBSs.

In particular, in most naturally occurring aromatic polyketide synthasegene clusters in actinomycetes, ORF1 and ORF2 are translationallycoupled. In order to facilitate construction of recombinant PKSs, theORF1 and ORF2 alleles used here were cloned as independent (uncoupled)cassettes. For act ORF1, the following sequence was engineered intopRM5: CCACCGGACGAACGCATCGATTAATTAAGGAGGACCATCATG, where the boldfacedsequence corresponds to upstream DNA from the acti region, TTAATTAA isthe PacI recognition site, and ATG is the start codon of act ORF1. Thefollowing sequence was engineered between act ORF1 and ORF2:NTGAATGCATGGAGGAGCCATCATG, where TGA and ATG are the stop and startcodons of ORF1 and ORF2, respectively, ATGCAT is the NsiI recognitionsite, and the replacement of N (A in act DNA, A or G in alleles fromother PKSs) with a C results in translational decoupling. The followingsequence was engineered downstream of act ORF2: TAATCTAGA, where TAA isthe stop codon, and TCTAGA is the XbaI recognition site. This allowedfusion of act ORF1 and ORF2 (engineered as above) to an XbaI site thathad been engineered upstream of act ORF3 (Khosla, C. et al. Molec.Microbiol. (1992) 6:3237). As a control, pRM2 was constructed, identicalto pRM5, but lacking any of the engineered sequences. ORF1 and ORF2 inpRM2 are translationally coupled. Comparison of the product profiles ofCH999/pRM2 and CH999/pRM5 revealed that the decoupling strategydescribed here had no detectable influence on product distribution orproduct levels.

EXAMPLE 3 Polyketides Produced using CH999 Transformed with pRM5

Plasmid pRM5 was introduced into S. coelicolor CH999 using standardtechniques. (See, e.g., Sambrook, et al. Molecular Cloning: A LaboratoryManual (Current Edition.) CH999 transformed with pRM5 produced a largeamount of yellowish-brown material. The two most abundant products werecharacterized by NMR and mass spectroscopy as aloesaponarin II (2)(Bartel, P. L. et al. J. Bacteriol. (1990) 172:4816) and itscarboxylated analog, 3,8-dihydroxy-1-methylanthraquinone-2-carboxylicacid (1) (Cameron, D. W. et al. Liebigs Ann. Chem. (1989) 7:699) (FIG.4). It is presumed that 2 is derived from 1 by non-enzymaticdecarboxylation (Bartel, P. L. et al. J. Bacteriol. (1990) 172:4816).Compounds 1 and 2 were present in approximately a 1:5 molar ratio.Approximately 100 mg of the mixture could be easily purified from 1 l ofculture. The CH999/pRM5 host-vector system was therefore functioning asexpected to produce significant amounts of a stable, only minimallymodified polyketide metabolite. The production of 1 and 2 is consistentwith the proposed pathway of actinorhodin biosynthesis (Bartel, P. L. etal. J. Bacteriol. (1990) 172:4816). Both metabolites, like theactinorhodin backbone, are derived from a 16-carbon polyketide with asingle ketoreduction at C-9.

When CH999 was transformed with pSEK4, identical to pRM5 except forreplacement of a 140 bp SphI/SalI fragment within the act KR gene by theSphI/SalI fragment from pUCl9, the resulting strain produced abundantquantities of the aromatic polyketide SEK4 (12). The exact structure ofthis product is slightly different from desoxyerythrolaccin (Bartel, P.L. et al. J. Bacteriol. (1990) 172:4816). However, in vivo isotopiclabeling studies using 1,2-¹³C₂- labeled acetate confirmed that thepolyketide backbone is derived from 8 acetates. Moreover, the aromaticregion of the ¹H spectrum, as well as the ¹³C NMR spectrum of thisproduct, are consistent with a tricyclic structure similar to 1, butlacking any ketoreduction (see Table 4).

EXAMPLE 4 Construction and Analysis of Hybrid Polyketide Synthases

A. Construction of Hybrid PKSs Including Components from act. gra andtcm PKSs

FIG. 1 shows the PKSs responsible for synthesizing the carbon chainbackbones of actinorhodin (3), granaticin (4), and tetracenomycin (5)(structures shown in FIG. 5) which contain homologous putative KS/AT andACP subunits, as well as the ORF2 product. The act and gra PKSs alsohave KRs, lacking in the tcm PKS. Corresponding proteins from eachcluster show a high degree of sequence identity. The percentageidentities between corresponding PKS proteins in the three clusters areas follows: KS/AT: act/gra 76, act/tcm 64, gra/tcm 70; CLF: act/gra 60,act/tcm 58, gra/tcm 54; ACP: act/gra 60, act/tcm 43, gra/tcm 44. The actand gra PKSs synthesize identical 16-carbon backbones derived from 8acetate residues with a ketoreduction at C-9 (FIG. 6). In contrast, alsoas shown in FIG. 6, the tcm polyketide backbone differs in overallcarbon chain length (20 instead of 16 carbons), lack of anyketoreduction, and regiospecificity of the first cyclization, whichoccurs between carbons 9 and 14, instead of carbons 7 and 12 for act andgra.

In an attempt to generate novel polyketides, differing in a range ofproperties, as well as to elucidate aspects of the programming ofaromatic PKSs, a systematic series of minimal PKS gene clusters, usingvarious permutations of the ORF1 (encoding the KS/AT subunit), ORF2(encoding the CLF subunit) and ORF3 (encoding the ACP subunit) geneproducts from the act, gra and tcm gene clusters were cloned into pRM5in place of the existing act genes, as shown in Table 1. The resultingplasmids were used to transform CH999 as above.

Analysis of the products of the recombinant PKSs containing variouspermutations among the KS/AT, ORF2 product, and ACP subunits of the PKSs(all constructs also containing the act KR, cyclase, and dehydratasegenes) indicated that the synthases could be grouped into threecategories (Table 1): those that did not produce any polyketide; thosethat produced compound 1 (in addition to a small amount of 2); and thosethat produced a novel polyketide 9 (designated RM20) (FIG. 6). Thestructure of 9 suggests that the polyketide backbone precursor of thismolecule is derived from 10 acetate residues with a single ketoreductionat the C-9 position.

In order to investigate the influence of the act KR on the reduction andcyclization patterns of a heterologous polyketide chain, pSEK15 was alsoconstructed, which included tcm ORFs 1-3, but lacked the act KR. (Thedeletion in the act KR gene in this construct was identical to that inpSEK4.) Analysis of CH999/pSEK15 showed the 20 carbon chain product,SEK15 (13) which resembled, but was not identical to, tetracenomycin Cor its shunt products. NMR spectroscopy was also consistent with acompletely unreduced decaketide backbone (see Table 4).

All act/gra hybrids produced compound 1, consistent with the identicalstructures of the presumed actinorhodin and granaticin polyketides. Ineach case where a product could be isolated from a tcm/act hybrid, thechain length of the polyketide was identical to that of the naturalproduct corresponding to the source of ORF2. This implies that the ORF2product, and not the ACP or KS/AT, controls carbon chain length.Furthermore, since all polyketides produced by the hybrids describedhere, except the ones lacking the KR (CH999/pSEK4 and CH999/pSEK15),underwent a single ketoreduction, it can be concluded that: (i) the KRis both necessary and sufficient for ketoreduction to occur; (ii) thisreduction always occurs at the C-9 position in the final polyketidebackbone (counting from the carboxyl end of the chain); and (iii) whileunreduced polyketides may undergo alternative cyclization patterns, innascent polyketide chains that have undergone ketoreduction, theregiochemistry of the first cyclization is dictated by the position ofthe resulting hydroxyl, irrespective of how this cyclization occurs inthe non-reduced product. In other words, the tcm PKS could be engineeredto exhibit new cyclization specificity by including a ketoreductase.

A striking feature of RM20 (9) is the pattern of cyclizations followingthe first cyclization. Isolation of mutactin (6) from an actVII mutantsuggested that the actVII product and its tcm homolog catalyze thecyclization of the second ring in the biosynthesis of actinorhodin (3)and tetracenomycin (5), respectively (Sherman, D. H. et al. Tetrahedron(1991) 47:6029; Summers, R. G. et al. J. Bacteriol. (1992) 174:1810).The cyclization pattern of RM20 (9) is different from that of 1 andtetracenomycin F1, despite the presence of the actVII gene on pRM20 (9).It therefore appears that the act cyclase cannot cyclize longerpolyketide chains.

Unexpectedly, the strain containing the minimal tcm PKS alone(CH999/pSEK33) produced two polyketides, SEK15 (13) and SEK15b (16), asdepicted in FIG. 8, in approximately equal quantities. Compounds (13)and (16) were also isolated from CH999/pSEK15, however, greaterquantities of compound (13) were isolated this construct than ofcompound (16).

SEK15b is a novel compound, the structure of which was elucidatedthrough a combination of NMR spectroscopy, sodium [1,2-¹³C₂] acetatefeeding experiments and mass spectroscopy. Results from ¹H and ¹³C NMRindicated that SEK15b consisted of an unreduced anthraquinone moiety anda pyrone moiety. Sodium [1,2-¹³C₂]-acetate feeding experiments confirmedthat the carbon chain of SEK15b was derived from 10 acetate units. Thecoupling constants calculated from the ¹³C NMR spectrum of the enrichedSEK15b sample facilitated peak assignment. Fast atom bombardment (FAB)mass spectroscopy gave a molecular weight of 381 (M+H⁺), consistent withC₂₀H₁₂O₈. Deuterium exchange was used to confirm the presence of eachhydroxyl in SEK15b.

In order to identify the degrees of freedom available in vivo to anascent polyketide chain for cyclizing in the absence of an activecyclase, polyketides produced by recombinant S. coelicolor CH999/pRM37(McDaniel et al. (1993), supra) were analyzed. The biosynthetic enzymesencoded by pRM37 are the tcm ketosynthase/acyltransferase (KS/AT), thetcm chain length determining factor (CLF), the tcm acyl carrier protein(ACP), and the act ketoreductase (KR).

Two novel compounds, RM20b (14) and RM20c (15) (FIG. 8) were discoveredin the culture medium of CH999/pRM37, which had previously yielded RM20(9). The relative quantities of the three compounds recovered were 3:7:1(RM20:RM20b:RM20c). The structures of (14) and (15) were elucidatedthrough a combination of mass spectroscopy, NMR spectroscopy and isotopelabeling experiments. ¹H and ¹³C NMR spectra suggested that RM20b andRM20c were diastereomers, each containing a pyrone moiety. Opticalrotations ([α]_(D) ²⁰ were found to by +210.8° for RM20b (EtOH, 0.55%)and +78.0° for RM20c (EtOH, 0.33%). Sodium [1,2-¹³C₂]-acetate feedingexperiments confirmed that the carbon chain of RM20b (and by inferenceRM20c) was derived from 10 acetate units. Deuterium exchange studieswere carried out in order to identify ¹H NMR peaks corresponding topotential hydroxyl groups on both RM20b and RM20c. Proton coupling.constants were calculated from the results of ¹H NMR and one-dimensionaldecoupling experiments. In particular, the coupling pattern in theupfield region of the spectrum indicated a 5-proton spin system of twomethylene groups surrounding a central carbinol methine proton. Highresolution fast atom bombardment (FAB) mass spectroscopy gave molecularweights of (519.0056) (M=Cs⁺) for RM20b and 387.1070 (M+H⁺) for RM20c,which is consistent with C₂₀H₁₈O₈ (M+Cs⁺, 519.0056; M+H⁺, 387.1080).Based on theses data, structures (14) and (15) (FIG. 8) were assigned toRM20b and RM20c, respectively.

Data from ¹H and ¹³C NMR indicated that the coupling constants betweenH-9 and the geminal protons on C-8 were 12.1 or 12.2 and 2.5 or 2.2 Hzfor RM20b or RM20c, respectively. The coupling constants between H-9 andthe geminal protons on C-10 were 9.6 or 9.7 and 5.7 or 5.8 Hz for Rm20bor RM20c, respectively. These values are typical of a J_(a,a) (J_(9a,8a)or J_(9a,10a)) and J_(a,e) (J_(9a,8e) or J_(9a,10e)) coupling pattern,and indicate an axial position for H-9 in both RM20b and Rm20c. Incontrast, the chemical shifts of the C-7 hydroxyls on the two moleculeswere 16.18 and 6.14 ppm for RM20b and RM20c, respectively. These valuesindicate a hydrogen bond between the C-7 hydroxyl and a suitablypositioned acceptor atom in RM20b, but not in RM20c. The most likelycandidate acceptor atoms for such hydrogen bonding are the C-13 carbonyloxygen in the conjugated pyrone ring system, or the bridge oxygen in theisolate pyrone ring. The former appears to be likely as it would beimpossible to discriminate between (14) and (15) if the latter were thecase. Furthermore, comparison of ¹³C NMR spectra of RM20b and RM20crevealed that the greatest differences between (14) and (15) were in thechemical shifts of the carbons that make up the conjugated pyrone ring(+5.9, −6.1, +8.9, −7.8 and +2.0 ppm for C-11, C-12, C-13, C-14 andC-15, respectively). Such a pattern of alternating upfield and downfieldshifts can be explained by the fact that the C-7 hydroxyl ishydrogen-bonded to the C-13 carbonyl, since hydrogen bonding would beexpected to reduce the electron density around C-11, C-13 and C-15, butincrease the electron density around C-12 and C-14. To confirm theC-7/C-13 hydrogen bond assignment, the exchangeable protons RM20b andRM20c were replaced with deuterium (by incubating in the presence ofD₂O), and the samples were analyzed by ¹³C NMR. The C-13 peak in RM20b,but not RM20c, underwent an upfield shift (1.7 ppm), which can beexplained by a weaker C-7/C-13 non-covalent bond in RM20b when hydrogenis replace with deuterium. In order to form a hydrogen bond with theC-13 carbonyl, the C-7 hydroxyl of RM20b must occupy the equatorialposition. Thus, it can be inferred that the C-7 and C-9 hydroxyls are onthe same face (syn) of the conjugated ring system in the major isomer(RM20b), whereas they are on opposite sides (anti) in the minor isomer(RM20c).

No polyketide could be detected in CH999/pRM15, /pRM35, and /pRM36.Thus, only some ORF1-ORF2 combinations are functional. Since eachsubunit was functional in at least one recombinant synthase, proteinexpression/folding problems are unlikely to be the cause. Instead,imperfect or inhibitory association between the different subunits ofthese enzyme complexes, or biosynthesis of (aborted) short chainproducts that are rapidly degraded, are plausible explanations.

B. Construction of Hybrid PKSs Including Components from act and frenPKSs

Streptomyces roseofulvus produces both frenolicin B (7) (Iwai, Y. et al.J. Antibiot. (1978) 31:959) and nanaomycin A (8) (Tsuzuki, K. et al. J.Antibiot. (1986) 39:1343). A 10 kb DNA fragment (referred to as the frenlocus hereafter) was cloned from a genomic library of S. roseofulvus(Bibb, M. J. et al. submitted) using DNA encoding the KS/AT and KRcomponents of the act PKS of S. coelicolor A3(2) as a probe (Malpartida,F. et al. Nature (1987) 325:818). (See FIG. 7 for structuralrepresentations.) DNA sequencing of the fren locus revealed theexistence of (among others) genes with a high degree of identity tothose encoding the act KS/AT, CLF, ACP, KR, and cyclase.

To produce the novel polyketides, the ORF1, 2 and 3 act genes present inpRM5 were replaced with the corresponding fren genes, as shown in Table2. S. coelicolor CH999, constructed as described above, was transformedwith these plasmids. (The genes encoding the act KR, and the act cyclasewere also present on each of these genetic constructs.) Based on resultsfrom similar experiments with act and tcm PKSs, described above, it wasexpected that the act KR would be able to reduce the products of allfunctional recombinant PKSS, whereas the ability of the act cyclase tocatalyze the second cyclization would depend upon the chain length ofthe product of the fren PKS.

The results summarized in Table 2 indicate that most of thetransformants expressed functional PKSs, as assayed by their ability toproduce aromatic polyketides. Structural analysis of the major productsrevealed that the producer strains could be grouped into two categories:those that synthesized compound 1 (together with a smaller amount of itsdecarboxylated side-product (2), and those that synthesized a mixture ofcompounds 1, 10 and 11 in a roughly 1:2:2 ratio. (Small amounts of 2were also found in all strains producing 1.) Compounds 1 and 2 had beenobserved before as natural products, and were the metabolites producedby a PKS consisting entirely of act subunits, as described in Example 3.Compounds 10 and 11 (designated RM18 and RM18b, respectively) are novelstructures whose chemical synthesis or isolation as natural products hasnot been reported previously.

The structures of 10 and 11 were elucidated through a combination ofmass spectroscopy, NMR spectroscopy, and isotope labeling experiments.The ¹H and ¹³C spectral assignments are shown in Table 3, along with¹³C-¹³C coupling constants for 10 obtained through sodium [1,2-¹³C₂)acetate feeding experiments (described below). Unequivocal assignmentsfor compound 10 were established with ID nuclear Overhauser effect (NOE)and long range heteronuclear correlation (HETCOR) studies. Deuteriumexchange confirmed the presence of hydroxyls at C-15 of compound 10 andC-13 of compound 11. Field desorption mass spectrometry (FD-MS) of 2revealed a molecular weight of 282, consistent with C₁₇H₁₄O₄ (282.2952).

Earlier studies showed that the polyketide backbone of 2 (Bartel, P. L.et al. J. Bacteriol. (1990) 172:4816) (and by inference, 1) is derivedfrom iterative condensations of 8 acetate residues with a singleketoreduction at C-9. It may also be argued that nanaomycin (8) arisesfrom an identical carbon chain backbone. Therefore, it is very likelythat nanaomycin is a product of the fren PKS genes in S. roseofulvus.Regiospecificity of the first cyclization leading to the formation of 1is guided by the position of the ketoreduction, whereas that of thesecond cyclization is controlled by the act cyclase (Zhang, H. L. et al.J. Org. Chem. (1990) 55:1682).

In order to trace the carbon chain backbone of RM18 (10), in vivofeeding experiments using (1,2-¹³C₂] acetate were performed onCH999/pRM18, followed by NMR analysis of labelled RM18 (10). The ¹³Ccoupling data (summarized in Table 3) indicate that the polyketidebackbone of RM18 (10) is derived from 9 acetate residues, followed by aterminal decarboxylation (the C-2 ¹³C resonance appears as an enhancedsinglet), which presumably occurs non-enzymatically. Furthermore, theabsence of a hydroxyl group at the C-9 position suggests that aketoreduction occurs at this carbon. Since these two features would beexpected to occur in the putative frenolicin (7) backbone, the resultssuggest that, in addition to synthesizing nanaomycin, the fren PKS genesare responsible for the biosynthesis of frenolicin in S. roseofulvus.This appears to be the first unambiguous case of a PKS with relaxedchain length specificity. However, unlike the putative backbone offrenolicin, the C-17 carbonyl of RM18 (10) is not reduced. This couldeither reflect the absence from pRM18 of a specific ketoreductase,dehydratase, and an enoylreductase (present in the fren gene cluster inS. roseofulvus), or it could reflect a different origin for carbons15-18 in frenolicin.

Regiospecificity of the first cyclization leading to the formation ofRM18 (10) is guided by the position of the ketoreduction; however thesecond cyclization occurs differently from that in 7 or 1, and issimilar to the cyclization pattern observed in RM20 (9), a decaketideproduced by the tcm PKS, as described above. Therefore, as in the caseof RM20 (9), it could be argued that the act cyclase cannot catalyze thesecond cyclization of the RM18 precursor, and that its subsequentcyclizations, which presumably occur non-enzymatically, are dictated bytemporal differences in release of different portions of the nascentpolyketide chain into an aqueous environment. In view of the ability ofCH999/pRM18 (and CH999/pRM34) to produce 1, one can rule out thepossibility that the cyclase cannot associate with the fren PKS (KS/AT,CLF, and ACP). A more likely explanation is that the act cyclase cannotrecognize substrates of altered chain lengths. This would also beconsistent with the putative biosynthetic scheme for RM20 (9).

A comparison of the product profiles of the hybrid synthases reported inTable 2 with analogous hybrids between act and tcm PKS components(Table 1) support the hypothesis that the ORF2 product is the chainlength determining factor (CLF). Preparation of compounds 9, 10 and 11via cyclization of enzyme-bound ketides is schematically illustrated inFIG. 8.

EXAMPLE 5 Construction and Analysis of Modular Polyketide Synthases

Expression plasmids containing recombinant modular DEBS PKS genes wereconstructed by transferring DNA incrementally from atemperature-sensitive “donor” plasmid, i.e., a plasmid capable ofreplication at a first, permissive temperature and incapable ofreplication at a second, non-permissive temperature, to a “recipient”shuttle vector via a double recombination event, as depicted in FIG. 10.pCK7 (FIG. 11), a shuttle plasmid containing the complete eryA genes,which were originally cloned from pS1 (Tuan et al. (1990) Gene 90:21),was constructed as follows. A 25.6 kb SphI fragment from pS1 wasinserted into the SphI site of pMAK705 (Hamilton et al. (1989) J.Bacteriol. 171:4617) to give pCK6 (Cm^(R)), a donor plasmid containingeryAII, eryAIII, and the 3′ end of eryAI. Replication of thistemperature-sensitive pSC101 derivative occurs at 30° C. but is arrestedat 44° C. The recipient plasmid, pCK5 (Ap^(R), Tc^(R)), includes a12.2kb eryA fragment from the eryAI start codon (Caffrey et al. (1992)FEBS Lett. 304:225) to the XcmI site near the beginning of eryAII, a 1.4kb EcoRI-BsmI pBR322 fragment encoding the tetracycline resistance gene(Tc), and a 4.0 kb NotI-EcoRI fragment from the end of eryAIII. PacI,NdeI, and ribosome binding sites were engineered at the eryAI startcodon in pCK5. pCK5 is a derivative of pRM5 (McDaniel et al. (1993),supra). The 5′ and 3′ regions of homology (FIG. 10, striped and unshadedareas) are 4.1 kb and 4.0 kb, respectively. MC1061 E. coli wastransformed (see, Sambrook et al., supra) with pCK5 and pCK6 andsubjected to carbenicillin and chloramphenicol selection at 30° C.Colonies harboring both plasmids (AP^(R), Cm^(R)) were then restreakedat 44° C. on carbenicillin and chloramphenicol plates. Only cointegratesformed by a single recombination event between the two plasmids wereviable. Surviving colonies were propagated at 30° C. under carbenicillinselection, forcing the resolution of the cointegrates via a secondrecombination event. To enrich for pCK7 recombinants, colonies wererestreaked again on carbenicillin plates at 44° C. Approximately 20% ofthe resulting colonies displayed the desired phenotype (Ap^(R),Tc^(S),Cm^(S)). The final pCK7 candidates were thoroughly checked viarestriction mapping. A control plasmid, pCK7f, which contains aframeshift error in eryAI, was constructed in a similar manner. pCK7 andpCK7f were transformed into E. coli ET12567 (MacNeil (1988) J.Bacteriol. 170:5607) to generate unmethylated plasmid DNA andsubsequently moved into Streptomyces coelicolor CH999 using standardprotocols (Hopwood et al. (1985) Genetic manipulation of Streptomyces. Alaboratory manual. The John Innes Foundation: Norwich).

Upon growth of CH999/pCK7 on R2YE medium, the organism produced abundantquantities of two polyketides (FIG. X). The addition of propionate (300mg/L) to the growth medium resulted in approximately a two-fold increasein yield of polyketide product. Proton and ¹³C NMR spectroscopy, inconjunction with propionic-1-¹³C acid feeding experiments, confirmed themajor product as 6dEB (17) (>40 mg/L). The minor product was identifiedas 8,8a-deoxyoleandolide (18) (>10 mg/L), which apparently originatesfrom an acetate starter unit instead of propionate in the 6dEBbiosynthetic pathway. ¹³C₂ sodium acetate feeding experiments confirmedthe incorporation of acetate into (18). Three high molecular weightproteins (>200 kDa), presumably DEBS1, DEBS2, and DEBS3 (Caffrey et al.(1992) FEBS Lett. 304:225), were also observed in crude extracts ofCH999/pCK7 via SDS-polyacrylamide gel electrophoresis. No polyketideproducts were observed from CH999/pCK7f.

Thus, novel polyketides, as well as methods for recombinantly producingthe polyketides, are disclosed. Although preferred embodiments of thesubject invention have been described in some detail, it is understoodthat obvious variations can be made without departing from the spiritand the scope of the invention as defined by the appended claims.

TABLE 1 Backbone ORF1 ORF2 ORF3 Major Carbon Plasmid (KS/AT) (CLDF)(ACP) Product(s) Length pRM5  act act act 1, 2 16 pRM7  gra act act 1, 216 pRM12 act gra act 1, 2 16 pRM22 act act gra 1, 2 16 pRM10 tcm act act1, 2 16 pRM15 act tcm act NP — pRM20 tcm tcm act 9 20 pRM25 act act tcm1, 2 16 pRM35 tcm act tcm NP — pRM36 act tcm tcm NP — pRM37 tcm tcm tcm9, 14, 15 20 pSEK15 tcm tcm tcm 13, 16 20 pSEK33 tcm tcm act 13, 16 20

TABLE 2 ORF1 ORF2 ORF3 Major Plasmid (KS/AT) (CDLF) (ACP) Product(s)pRM5  act act act 1, 2 pRM8  fren act act 1, 2 pRM13 act fren act NPpRM23 act act fren 1, 2 pRM18 fren fren act 1, 2, 10, 11 pRM32 fren actfren NP pRM33 act fren fren NP pRM34 fren fren fren 0001, 2, 10, 11

TABLE 3 ¹H(400 MHz) and ¹³C(100 MHz) NMR data from RM18(10) andRM18b(11) RM18 RM18b ¹Hδ(ppm) ¹Hδ(ppm) carbon^(α) ¹³Cδ(ppm) (J_(CC)(Hz))(m, J_(HH)(Hz), area)) carbon^(α) ¹³Cδ(ppm) (m, J_(HH)(Hz), area)) 229.6 NC^(b) 2.2(s, 3H) 3 203.7 37.7 4 47.0 36.9 3.6(s, 2H) 2 18.8 2.1(s,3H) 5 149.6 77.2 3 152.3 6 106.7 77.4 6.2(s, 1H) 4 104.0 6.1(s, 1H) 7129.1 61.9 5 130.0 8 114.4 62.1 6.7(d, 7.2, 1H) 6 113.5 6.7(d, 7.0) 9130.1 58.9 7.3(dd, 8.4, 7.4, 1H) 7 130.1 7.3(dd, 7.1, 8.7, 1H) 10 120.659.2 7.6(d, 8.9, 1H) 8 120.1 7.6(d, 8.6, 1H) 11 132.7 56.0 9 132.8 12116.7 55.7 10 116.6 13 155.6 74.7 11 155.9 14 98.4 74.9 12 98.2 15 158.869.6 6.4(s, 1H) 13 159.1 6.4(s, 1H) 16 113.6 69.3 11.2(s, 1OH) 14 113.811.2(s, 1OH) 17 201.7 41.9 15 201.7 18 32.4 41.7 2.5(s, 3H) 16 32.42.5(s, 3H) ^(α)carbons are labelled according to their number in thepolyketide backbone ^(b)NC, not coupled

TABLE 4 ¹H and ¹²C NMR data for SEK4 (12) and SEK15 (13)^(a) SEK4 (12)SEK15 (13) carbon^(b) ¹³Cδ(ppm) J_(CC)(Hz) ¹Hδ(ppm) carbon ¹³Cδ(ppm)J_(CC)(Hz) ¹Hδ(ppm) 1 165.4 78.8 11.60(s, 1OH) 1 164.0 79.1 12.20(s,1OH) 2 88.2 79.8 6.26(d, J = 2.28 Hz, 1H) 2 88.2 79.4 6.20(d, J = 1.88Hz, 1H) 3 170.5 55.3 3 172.8 57.9 4 111.3 61.3 6.33(d, J = 2.24 Hz, 1H)4 101.8 53.9 6.20(d, J = 1.88 Hz, 1H) 5 163.8 51.0 5 163.1 50.4 6 37.650.8 4.07(d, J = 15.7 Hz, 1H) 6 36.7 50.8 1.90(s, 2H) 4.16(d, J = 16.0Hz, 1H) 7 138.6 60.7 7 135.4 60.7 8 102.9 60.9 5.66(d, J = 1.6 Hz, 1H) 8109.1 61.7 5.66(s, 1H) 9 161.9 71.9 10.50(s, 1OH) 9 159.8 66.2 10 100.670.9 5.19(d, J = 1.96 Hz, 1H) 10 101.6 66.5 5.08 (s, 1H) 11 162.9 60.811 157.4 67.3 12 112.9 61.6 12 121.1 67.6 13 191.1 39.1 13 200.3 58.1 1449.3 39.9 2.54(d, J = 15.9 Hz, 1H) 14 117.2 58.6 4.92(d, J = 16.0 Hz,1H) 15 99.6 46.6 6.90(s, 1OH) 15 163.6 68.5 16 27.5 46.8 1.56(s, 3H) 16100.6 68.0 6.08 (s, 1H) 17 162.2 62.6 18 111.0 62.0 6.12 (s, 1H) 19141.9 43.3 20 21.1 42.7 1.86 (s, 3H) ^(a) ¹H and ¹³C NMR's were recordedin DMSO-d₆ (400 MHz for ¹1H and 100 MHz for ¹³C) ^(b)carbons arelabelled according to their number in the polyketide backbone

2 42 base pairs nucleic acid single linear DNA (genomic) 1 CCACCGGACGAACGCATCGA TTAATTAAGG AGGACCATCA TG 42 25 base pairs nucleic acid singlelinear DNA (genomic) 2 NTGAATGCAT GGAGGAGCCA TCATG 25

What is claimed is:
 1. A method to prepare a cell containing at leastone nucleic acid molecule, wherein said molecule comprises at least onemodule that encodes a modular polyketide synthase (PKS) functional incatalyzing the synthesis of a polyketide, said module comprising atleast one nucleotide sequence which encodes a PKS acyl transferase (AT)activity; at least one nucleotide sequence which encodes a PKS ketoacylcarrier protein synthase (KS) activity; and at least one nucleotidesequence which encodes a PKS acyl carrier protein (ACP) activity;wherein said module is a 6-deoxyerythronolide B synthase module; saidmodule operatively linked to a control sequence, whereby a functionalmodular PKS is produced in said cell, with the proviso that said moduleor said control sequence is heterologous to the host cell; said methodcomprising introducing said nucleic acid molecule into a host cell. 2.The method of claim 1 wherein said host cell has been modified so ascompletely to lack a PKS gene cluster normally present in the unmodifiedsaid host cell.
 3. The method of claim 2 wherein said host cell is S.coelicolor.
 4. The method of claim 1 wherein said module comprises: atleast one nucleotide sequence encoding PKS ketoteductase (KR) activity;or at least one nucleotide sequence encoding PKS ketoreductase (KR)activity and at least one nucleotide sequence encoding PKS dehydratase(DH) activity; or at least one nucleotide sequence encoding PKSketoreductase (KR) activity and at least one nucleotide sequenceencoding PKS dehydratase (DH) activity, and at least one nucleotidesequence encoding PKS enoyl reductase (ER) activity; and optionally, atleast one nucleotide sequence encoding PKS thioesterase (TE) activity.5. The method of claim 4 wherein said module comprises: at least onenucleotide sequence encoding PKS ketoreductase (KR) activity and atleast one nucleotide sequence encoding PKS dehydratase (DH) activity;and at least one nucleotide sequence encoding PKS enoyl reductase (ER)activity.
 6. The method of claim 1 wherein said nucleic acid moleculeand optionally additional nucleic acid molecules comprise the complete6-deoxyerythronolide B synthase gene cluster.
 7. A method to produce afunctional polyketide synthase which method comprises culturing cellsprepared by the method of claim 1 under conditions wherein said moduleis expressed.
 8. A method to produce a functional polyketide synthasewhich method comprises culturing cells prepared by the method of claim 4under conditions wherein said module is expressed.
 9. A method toproduce a functional polyketide synthase which method comprisesculturing cells prepared by the method of claim 6 under conditionswherein said module is expressed.
 10. The method of claim 1 wherein thehost cell is an Actinomycete.
 11. The method of claim 10 wherein thehost cell is a Streptomyces.
 12. The method of claim 11 wherein the hostcell is S. coelicolor or S. lividans.
 13. The method of claim 7 whereinsaid cells are Actinomycetes.
 14. The method of claim 13 wherein saidcells are Streptomyces.
 15. The method of claim 14 wherein said cellsare S. coelicolor or S. lividans.